概要: Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here, we performed single-cell RNA-seq analysis of over 2000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that the female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis and cell cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of BMP and Notch signaling pathways. Our work provides key insights into crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo.
项目整体设计: Here we performed single cell RNA-seq analysis for 2,167 individual PGCs as well as their gonadal niche cells, covering the developmental stages from 4 to 26 weeks after gestation in both female and male human embryos
A modified Smart-seq2 protocol was applied for single-cell RNA-seq. Briefly, after MACS and FACS purification, a single PGC or gonadal somatic cell was picked into the lysis buffer by mouth pippete. The reverse transcription reaction was performed with 24 oligo (dT) primer anchored with the 8 bp cell specific barcode, and also with 8 bp unique molecular identifiers (UMIs).
建库方案:
After the first-strand reaction, the cDNA was amplified by 17 cycles. Then the amplified cDNA of the PGCs of the same fetal sample were pooled together and the amplified cDNA of the gonadal somatic cells of the same fetal sample were pooled together to proceed the following steps. Biotinylated pre-indexed primer was used to amplify the PCR product by additional 4 cycles to introduce biotin tags to the 3'end of the amplified cDNA. About 300 ng DNA was sheared to around 300 bp by Covaris S2 and the 3'ternimal of the DNA was captured by Dynabeads® MyOne™Streptavidin C1 beads (Thermo Fisher). RNA seq library was constructed using Kapa Hyper Prep Kit (Kapabiosystems) and subjected to 150 bp paired-end sequencing on illumina HiSeq 2500 platform (sequenced by Novogene).
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
paired
库的链类型:
Reverse; Forward; -
测序平台:
Illumina
测序仪型号:
Illumina HiSeq 2500
链特异性:
Specific; Unspecific
样本
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文章
Single-Cell RNA-Seq Analysis Maps Development of Human Germline Cells and Gonadal Niche Interactions.
